Anti-inflammatory polypeptides

ABSTRACT

This invention concerns anti-inflammatory agents, compositions, and methods for treating inflammatory disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 14/368,701, filed on Jun. 25, 2014, which is a U.S.National Phase based on International Application No. PCT/US2012/068718,filed on Dec. 10, 2012, which claims priority to U.S. ProvisionalApplication No. 61/577,361, filed on Dec. 19, 2011. The contents of theprior applications are incorporated herein by reference in theirentireties.

FIELD OF INVENTION

This invention relates to anti-inflammatory agents, compositions, andmethods for treating inflammatory disorders.

BACKGROUND

Inflammatory disorders, including autoimmune diseases, are disordersinvolving abnormal activation and subsequent migration of white bloodcells to affected areas of the body. These conditions encompass a widerange of ailments that affect the lives of millions of people throughoutthe world. Although various treatments are presently available, manypossess significantly side effects or are not very effective inalleviating all symptoms. Thus, there are needs for anti-inflammatoryagents for treating inflammatory disorders and needs for methods ofidentifying and evaluating such agents.

Immunoglobulin G (IgG) has long been appreciated to mediate both pro-and anti-inflammatory activities through interactions mediated by its Fcfragment. While Fc-FcγR interactions are responsible for thepro-inflammatory properties of immune complexes and cytotoxicantibodies, intravenous gamma globulin (IVIG) and its Fc fragments areanti-inflammatory and are widely used to suppress inflammatory diseases.It has been proposed that glycosylation of IgG is crucial for regulationof cytotoxicity and inflammatory potential of IgG. For example, it hasbeen suggested that anti-inflammatory activity of IVIG is a property ofthe Fc fragment and its linked glycan, requiring terminal α.2,6 sialicacid linkages, indicating a combined requirement for the specificpolypeptide backbone and glycan structure for immunosuppression.(Anthony, et al., 2008, Science 320: 373-376 and WO 2007/117505).

However, only a minor population of IgG in IVIG have glycans terminatingin α2,6 sialic acids (sFc) and the anti-inflammatory activity. As aresult, for the suppression of autoantibody triggered inflammation in avariety of clinical settings, one has to administer IVIG at high doses(1-2 g/kg), to enrich sialylated IgGs, or otherwise to increase thesialylation of IgGs (US Application Nos. 20080206246, and 20090004179,and Nimmerjahn et al. Annu Rev Immunol 26, 513-533 (2008)).

The present invention addresses and meets the above-mentioned needs byidentifying sialylation-free anti-inflammatory polypeptides.

SUMMARY

This invention relates to agents, such as polypeptides and antibodies,and methods for treating inflammatory disorders, e.g., autoimmunediseases.

Accordingly, one aspect of this invention features an isolatedpolypeptide comprising a modified sequence that is at least 75% (e.g.,any number between 75% and 100%, inclusive, e.g., 75%, 80%, 85%, 90%,95%, 99%, and 100%) identical to an IgG Fc region. The modified sequenceis free of sialylation and the polypeptide has an anti-inflammatoryactivity that is higher than that of a parent polypeptide. The parentpolypeptide can comprise the IgG Fc region, such as the sequence of SEQID NO: 1 listed below. In some embodiments, the polypeptide has abilityto bind to DC-SIGN, and to bind to hFcγRIIA or RIIB In one embodiment,the isolated polypeptide has an ability to bind to hFcγRIIA or RIIB at aK_(D) of 2×10⁻⁵ M or lower (i.e., K_(A) of 5.0×10⁴ M⁻¹ or higher).Preferably, the modified sequence has a FA241 mutation. The modifiedsequence can be at least 75% (e.g., any number between 75% and 100%,inclusive, e.g., 75%, 80%, 85%, 90%, 95%, 99%, and 100%) identical toSEQ ID NO: 2. In some examples, the modified sequence comprises orconsists essentially of SEQ ID NO: 2.

In another aspect, the invention provides a method for making apolypeptide having an anti-inflammatory activity. The method includes,among others, steps of providing a parent polypeptide having thesequence of an IgG Fc region or a first nucleic acid sequence encodingthe parent polypeptide; and modifying the parent polypeptide to obtain amodified polypeptide so that the modified polypeptide is free ofsialylation and mimics the structural of a sialylated form of the IgG Fcregion. The modifying step can be conducted by modifying the firstnucleic acid sequence to obtain a second nucleic acid encoding themodified polypeptide. The invention also provides a polypeptide made bythe just-described method.

In a third aspect, the invention features an isolated nucleic acidcomprising a sequence encoding the polypeptide described above; anexpression vector comprising the nucleic acid; and a host cellcomprising the nucleic acid. The invention also features a method ofproducing a polypeptide. The method includes culturing the host cell ina medium under conditions permitting expression of a polypeptide encodedby the nucleic acid, and purifying the polypeptide from the culturedcell or the medium of the cell.

In a fourth aspect, the invention features a pharmaceutical formulationcomprising (i) the polypeptide or nucleic acid described above, and (ii)a pharmaceutically acceptable carrier.

In a fifth aspect, the invention provides a method of treating aninflammatory disease. The method includes administering to a subject inneed thereof a therapeutically effective amount of the above-describedpolypeptide or nucleic acid encoding the polypeptide. Also provided isuse of the polypeptide or nucleic acid in the manufacture of amedicament for treating an inflammatory disease. The invention alsofeatures an isolated polypeptide, nucleic acid, expression vector, hostcell, composition, or method for treating an inflammatory diseasesubstantially as shown and described herein.

In another aspect, the invention further provides a method of increasinga level of regulatory T (T_(reg)) cells in a subject in need thereof.The method comprises administering to the subject an effective amount of(i) a first isolated polypeptide comprising an IgG Fc region that issialylated; or (ii) a second isolated polypeptide comprising a modifiedsequence that is at least 75% (e.g., any number between 75% and 100%,inclusive, e.g., 75%, 80%, 85%, 90%, 95%, 99%, and 100%) identical tothe IgG Fc region or a nucleic acid encoding the second polypeptide,wherein the modified sequence has a FA241 mutation. In one embodiment,the subject has an inflammatory disease, such as an autoimmune disease,including a T cell-mediated autoimmune disease. Examples of the Tcell-mediated autoimmune disease include multiple sclerosis and type Idiabetes. Accordingly, the invention also provides a method treating a Tcell-mediated autoimmune disease in a subject in need thereof. Themethod comprises administering to the subject an effective amount of theabove-mentioned first isolated polypeptide or second isolatedpolypeptide or nucleic acid.

In preferred embodiments, the IgG region in the first isolatedpolypeptide can be sialylated at a level higher than that of IgG in thesubject. The modified sequence can be sialylated at different levels ornon-sialylated. In some embodiments, it is (a) substantially free ofsialylation or (b) sialylated at a level lower than that of IgG of thesubject. The IgG Fc region can comprise the sequence of SEQ ID NO: 1.The modified sequence can be at least 75% (e.g., any number between 75%and 100%, inclusive, e.g., 75%, 80%, 85%, 90%, 95%, 99%, and 100%)identical to SEQ ID NO: 2. The first or second isolated polypeptide hasan ability to bind to DC-SIGN, hFcγRIIA, or hFcγRIIB. Preferably, theisolated polypeptide has an ability to bind to hFcγRIIA or hFcγRIIB at aK_(D) of 2×10⁻⁵ M or lower (i.e., K_(A) of 5.0×10⁴ M⁻¹ or higher).

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1D show that F241A is capable of suppressing serum-inducedarthritis. (a) Schematic depiction of the interaction between mannoseresidues of the non-sialylated core glycan (Asialo-Fc) with thephenylalanine residue at position 241 of the Cγ2 domain. Uponsialylation (Sial-Fc) this interaction no longer occurs, resulting in ahigher conformational flexibility of the Fc portion to switch betweenthe so-called open and closed conformation. (b) hDC-SIGN⁺ bonemarrow-derived macrophages were pulsed for 1 hour with sialylated (sFc)or non-sialylated (Fc) wild-type Fc or F241A-Fc. Whole cell extractswere used to analyze IL-33 production by western blotting. Actin servedas loading control. (c) BMMΦ from either SIGN-R1^(−/−) or hDC-SIGN⁺ micewere pulsed with non-sialylated wild-type Fc (asialo-Fc), sialylatedwild-type Fc (sFc) or F241A. After extensive washes the cells wereadoptively transferred into K/B×N challenged C57BL/6 mice. (d) hDC-SIGN⁺BMMΦ were pulsed with either asialylated (asialo Fc) or sialylated(α2,6sFc) variants of wild-type Fc or F241A. The cells were adoptivelytransferred into K/B×N challenged C57BL/6 mice. Means+/−SEM are plotted;*p<0.05; **p<0.01 determined by Tukey's post-hoc test.

FIGS. 2A-2C show that F241A induces anti-inflammatory responses throughengagement of type II Fc receptors. (a) C57BL/6 wild-type mice weretreated with PBS, IL-4ic or F241A (0.033 g/kg) and were challenged onehour later with K/B×N sera. Blood was collected from these mice 6 dayspost treatment and serum IL-6 levels were analyzed by ELISA. (b)Clinical scores of mice were monitored reflecting the severity ofserum-induced arthritis. (c) SIGN-R1^(−/−) and hDC-SIGN⁺ mice werechallenged with K/B×N serum and treated with sialylated wild-type Fc(sFc) or F241A (both 0.033 g/kg). Development of disease was monitoreddaily until day 6 post disease induction.

FIGS. 3A-3C show that sFc/F241A activates and expands T_(reg) cells. (a)C57BL/6 wild-type mice received intravenous injections of IVIG (1 g/kg),IVIG-F(ab′)₂ (0.66 g/kg), IVIG-Fc (0.33 g/kg) or PBS as control. On day5 post injection T_(reg) cell numbers in spleens were analyzed by flowcytometry. (b) K/B×N-challenged C57BL/6 wild-type mice were given IVIG(1 g/kg), neuraminidase-treated IVIG (NA-IVIG) (1 g/kg), non-sialylatedF241A (0.033 g/kg) or PBS as control. Clinical signs of arthritis weremonitored. (c) On day 5 post treatment mice were euthanized and T_(reg)cells in spleens were analyzed by flow cytometry. Means+/−SEM areplotted; *p<0.05; **p<0.01; ***p<0.001 determined by Tukey's post-hoetest.

FIGS. 4A-4F show that IVIG-/F241A-activated T_(reg) cells efficientlysuppress T cell-mediated autoimmunity in experimental autoimmuneencephalomyelitis (EAE) mice. (a) C57BL/6 wild-type mice were immunizedwith MOG₃₅₋₅₅ peptide to induce EAE. Starting on day five post EAEinduction mice received intravenous injections of PBS, IVIG (1 g/kg) orneuraminidase-treated IVIG (NA-IVIG) (1 g/kg) every 5 days. Clinicalscores of EAE are shown. (b,c) Mice were euthanized and CD4⁺ effector Tcells (b) and T_(reg) cells (c) from draining lymph nodes were analyzedby flow cytometry. (d) EAE was induced in C57BL/6 wild-type mice byimmunization with MOG₃₅₋₅₅ peptide. Every five days mice receivednon-sialylated F241A (0.033 g/kg) or PBS intravenously. For T_(reg) celldepletion mice were given the T_(reg) cell depletion antibody PC61 (400μg) 3 days before EAE induction as well as every fifth day postinduction. Clinical scores of disease are depicted. (e,f) Cells fromdraining lymph nodes of EAE mice were isolated and analyzed for thepercentages of CD4⁺ effector T cells (e) and T_(reg) cells (f) by flowcytometry. Means+/−SEM are plotted; *p<0.05; **p<0.01; ***p<0.001determined by Tukey's post-hoc test.

FIGS. 5A-5D show requirement of type II Fc receptors forIVIG-/F241A-mediated T_(reg) cell activation. EAE was induced in allexperiments by immunization with MOG₃₅₋₅₅ peptide. Mice receivedintravenous injections on days 5, 10, 15 and 20. Clinical scores of EAEare depicted. (a) C57BL/6 wild-type and SIGN-R1^(−/−) mice were treatedwith IVIG (1 g/kg) or PBS. (b) SIGN-R1^(−/−) mice were given IVIG (1g/kg) as mentioned above or IL-33 (0.5 μg) intraperitoneally every twodays starting on day five post EAE induction. (c) SIGN-R1^(−/−) andSIGN-R1^(−/−) hDC-SIGN⁺ mice were treated with PBS or IVIG (1 g/kg). (d)SIGN-R1^(−/−) hDC-SIGN⁺ mice received non-sialylated F241A(asialo-F241A) (0.033 g/kg) or PBS control. Means+/−SEM are plotted;*p<0.05; **p<0.01; ***p<0.001 determined by Tukey's post-hoc test.

FIGS. 6A-6G show that F241A-induced IL-33 production activates T_(reg)cells. (a,b) C57BL/6 wild-type mice were given IL-33 (0.5 μg)intraperitoneally on four consecutive days. On day five mice wereeuthanized and the percentages of splenic T_(reg) cells were analyzed byflow cytometry. (c) Bone marrow-derived macrophages from C57BL/6wild-type or hDC-SIGN mice were pulsed with PBS, IVIG or non-sialylatedF241A. (c) Total RNA was isolated and used for quantitative rtPCR tomeasure IL-33 mRNA levels. The housekeeping gene Gapdh was used fornormalization. (d, e) After treatment bone marrow-derived macrophageswere extensively washed and adoptively transferred into C57BL/6wild-type recipient mice. On day five post transfer mice were sacrificedand T_(reg) cells and ST2 expression were analyzed by flow cytometry.(f) EAE was induced in C57BL/6 wild-type mice by immunization withMOG₃₅₋₅₅ peptide. Every two days post induction mice received IL-33 (0.5μg i.p.). Clinical scores of disease are shown. (g) The percentages ofT_(reg) cells from draining lymph nodes of EAE mice were analyzed byflow cytometry. Means+/−SEM are plotted; ***p<0.001 determined byTukey's post-hoc test.

FIGS. 7A-7D show that IVIG/F241A induces T_(reg) cell activation bysignaling through the IL-33/ST2 axis. (a,b) K/B×N-challenged C57BL/6wild-type mice were treated with PBS or IVIG (1 g/kg). In order to blockIL-33 signaling mice received an anti-ST2 blocking antibody (100 μgi.v.) or isotype control. Clinical scores of RA are shown (a). On dayfive, post disease induction mice were sacrificed and splenic T_(reg)cells were analyzed by flow cytometry (b). (c) EAE was induced inC57BL/6 wild-type mice by immunization with MOG₃₅₋₅₅ peptide. Mice weretreated intravenously with PBS or non-sialylated F241A (asialo-F241A)(0.033 g/kg) four times on day 5, 10, 15 and 20. Each injection precededan intravenous injection of an anti-ST2 blocking antibody. Shown areclinical scores of EAE. (d) EAE mice were sacrificed and cells isolatedfrom draining lymph nodes. T_(reg) cells and their Foxp3 and ST2expression were analyzed by flow cytometry. Means+/−SEM are plotted;*p<0.05; ***p<0.001 determined by Tukey's post-hoe test.

FIG. 8 shows that F241A binds to DC-SIGN independent of sialylation.Recombinant DC-SIGN was immobilized and binding affinity of asialylated(asial F241A), sialylated F241A (sial F241A) and asialylated wild-typeFc (asial WT Fc) was measured by ELISA.

FIGS. 9A-9C show that the E318/Lys340 pocket is critical for theanti-inflammatory effect of sialylated Fc. (a) Bone marrow cells fromSIGN-R1^(−/−) and hDC-SIGN⁺ mice were isolated and differentiated invitro into macrophages (BMMΦ). BMMΦ were pulsed with wild-type or mutantFc overnight. Whole cell extracts were used for western blotting. Actinserved as loading control. (b) K/B×N-challenged C57BL/6 mice weretreated with different Fc preparations. Clinical scores of disease weremonitored on day six post treatment. (c) Serum IL-6 levels were testedby ELISA 8 days post treatment.

FIGS. 10A-10C: IVIG selectively expands inducible T_(reg) cells. EAE wasinduced in C57BL/6 wild-type mice by immunization with MOG₃₅₋₅₅ peptideemulsified in CFA. Mice were treated with PBS or IVIG (1 g/kg). (a)Clinical scores of EAE are depicted. Means+/−SEM are plotted; **p<0.01determined by Tukey's post-hoc test. (b) Representative images of HE andLuxol Fast Blue staining of spinal cords from EAE mice. HE staining forinflammation. Loss of signal in the Luxol Fast Blue staining reflectsprogressed demyelination. (c) Cells from draining lymph nodes wereisolated and analyzed for T_(reg) cell numbers and their expression ofthe nT_(reg)-specific transcription factor Helios.

FIGS. 11A-11B show that loss of SIGN-R1 abrogates the positive effect ofIVIG on T_(reg) cells. C57BL/6 wild-type and SIGN-R1^(−/−) mice weregiven IVIG (1 g/kg) or PBS intravenously. One hour later mice werechallenged with K/B×N serum. (a) Clinical signs of arthritic diseasewere monitored 5 days after challenge. (b) T_(reg) cells from spleenswere analyzed by flow cytometry. Means+/−SEM are plotted; **p<: 0.01determined by Student's t test.

FIG. 12 shows that IL-33 synergistically contributes to T_(reg) celldifferentiation in vitro. Naïve CD4⁺ T cells were isolated from spleensof C57BL/6 wild-type mice. Cells were cultured three days in presence ofanti-CD3/CD28. To drive T_(reg) cell differentiation TGF-β was added tothe cells either alone or in combinations with IL-33 and IL-23. T_(reg)cell numbers and ST2 expression were analyzed by flow cytometry.

FIGS. 13A-13C show that basophil depletion does not affect IVIG-mediatedT_(reg) cell stimulation. C57BL/6 wild-type mice were treated withanti-FcεRI antibody to deplete basophils or with an isotype control. Inaddition mice received IVIG (1 g/kg) or PBS and were challenged withK/B×N sera. (a) FACS analysis of splenic basophils five days afterinjection. (b) Clinical signs of arthritic disease were monitored on dayfive post disease induction. (c) Splenic T_(reg) cells were analyzed byflow cytometry. Means+/−SEM are plotted; *p<0.05, **p<0.01 determined byTukey's post-hoc test.

FIGS. 14A-14D show that VIG preferentially activates iT_(reg) cells andprotects from experimental colitis. C57BL/6 Rag1^(−/−) mice wereinjected with CD4⁺ CD45RB^(high)CD25⁻ T cells in order to induce T celltransfer colitis. Mice were treated once a week with PBS or IVIG (1g/kg) starting four weeks post T cell transfer. Rag1^(−/−) control mice(CTRL) did not receive any T cells. (a) Body weight was measured once aweek. Body weight loss was used as a means of disease severity. (b)Cells from draining lymph nodes were analyzed for percentages of T_(reg)cells and CD4⁺ effector T cells by FACS. (c) Entire colons weredissected and measured. Colon shrinkage reflected disease severity andcorrelated with body weight loss. (d) Representative image of dissectedcolons as described in c. Means+/−SEM are plotted; ***p<0.001 determinedby Tukey's post-hoe test.

FIG. 15 shows a model of sFc-induced anti-inflammatory pathways:Sialylated IgG, sFc as well as the sialylated Fc analogue F241Aselectively engage type II FcRs like SIGN-R1 or human DC-SIGN onregulatory macrophages and induce IL-33 production. IL-33 is a centralmediator that induces two different anti-inflammatory pathways.Basophils respond to IL-33 with production of IL-4, which in turninduces the up-regulation of the inhibitory FcγRIIB on effectormacrophages. The resulting dramatic change of their activation thresholdsuppresses inflammation. In addition IL-33 also triggers activation andexpansion of T_(reg) cells, which effectively suppress T_(H)1 andT_(H)17 cells, thus ameliorating T cell-mediated autoimmunity.

DETAILED DESCRIPTION

This invention is based, at least in part, on unexpected discoveriesthat non-sialylated IgG Fc variants confer anti-inflammatory activityand mimic the effect of 2,6 sialylated Fc as anti-inflammatory mediatorsand that structural alterations induced by sialylation can be mimickedby specific amino acid modifications to the C_(H)2 domain. It was alsounexpected that both the variants and sialylated Fc activates T_(reg)cells.

As disclosed herein, the anti-inflammatory activity of IVIG is dependenton the presence of sialic acid in the core IgG-Fc glycan, resulting inincreased conformational flexibility of the C_(H)2 domain, withcorresponding modulation of FcR binding specificity from type I to typeII receptors. Sialylated IgG-Fc (sFc) increases the activation thresholdof innate effector cells to immune complexes by stimulating theup-regulation of the inhibitory receptor FcγRIIB The inventors havefound that the structural alterations induced by sialylation can bemimicked by specific amino acid modifications to the C_(H)2 domain. Forexample, an IgG-Fc variant with a point mutation at position 241 (F-A)exhibits anti-inflammatory activity even in the absence of sialylation.F241A and sFc protect mice from arthritis in both collagen andKB×N-induced models and, in the T cell-mediated EAE mouse model,suppressed disease by specifically activating T_(reg) cells. Protectionby these anti-inflammatory Fcs in both antibody- and T cell-mediatedautoimmune diseases required type II FcRs and the induction of IL-33.These results further clarify the mechanism of action of IVIG in bothantibody and T cell-mediated inflammatory diseases and demonstrate thatFc variants that mimic the structural alterations induced bysialylation, such as F241A, can be used for the treatment of variousautoimmune disorders.

IgG and Fc Sialylation

IgG is the major serum immunoglobulin. It is a glycoprotein composed oftwo identical heavy chains and two light chains, which in turn arecomposed of variable and constant domain. IgG contains a single,N-linked glycan at Asn²⁹⁷ in the CH2 domain on each of its two heavychains. The covalently-linked, complex carbohydrate is composed of acore, biantennary penta-polysaccharide containing N-acetylglucosamine(GlcNAc) and mannose (man). Further modification of the corecarbohydrate structure is observed in serum antibodies with the presenceof fucose, branching GlcNAc, galactose (gal) and terminal sialic acid(sa) moieties variably found. Over 40 different glycoforms have thusbeen detected to be covalently attached to this single glycosylationsite (Fujii et al., J. Biol. Chem. 265, 6009, 1990). Glycosylation ofIgG has been shown to be essential for binding to all FcγRs bymaintaining an open conformation of the two heavy chains. Jefferis andLund, Immune. Lett. 82, 57 (2002), Sondermann et al., J. Mol. Biol. 309,737 (2001). It is believed that this IgG glycosylation for FcγR bindingaccounts for the inability of deglycosylated IgG antibodies to mediatein vivo triggered inflammatory responses, such as ADCC, phagocytosis andthe release of inflammatory mediators. Nimmerjahn and Ravetch, Immunity24, 19 (2006). Further observations that individual glycoforms of IgGmay contribute to modulating inflammatory responses has been suggestedby the altered affinities for individual FcγRs reported for IgGantibodies containing or lacking fucose and their consequential affectson cytotoxicity. Shields et al., J. Biol. Chem. 277, 26733 (2002),Nimmerjahn and Ravetch, Science 310, 1510 (2005). A link betweenautoimmune states and specific glycosylation patterns of IgG antibodieshas been observed in patients with rheumatoid arthritis and severalautoimmune vasculities in which decreased galactosylation andsialylation of IgG antibodies have been reported. Parekh et al., Nature316, 452 (1985), Rademacher et al., Proc. Natl. Acad. Sci. USA 91, 6123(1994), Matsumoto et al., 128, 621 (2000), Holland et al., Biochim.Biophys. Acta December 27. Variations in IgG glycoforms have also beenreported to be associated with aging and upon immunization, although thein vivo significance of these alterations has not been determined.Shikata et al., Glycoconj. J. 15, 683 (1998), Lastra, et al.,Autoimmunity 28, 25 (1998).

IgG Fc Variants

As disclosed herein, certain IgG Fc variants, sialylated or not,surprisingly also confer anti-inflammatory activity. Such variants,including the FA241 variant, represent species within a larger genus ofmolecules that, by virtue of mimicking the structural and biologicalproperties of sialylated Fc, but do not require sialylation, can bedeveloped as anti-inflammatory therapeutics.

IVIG, although initially developed as an immunoglobulin replacementtherapy in patients with hypogammagloubilinenemia, has gained widespreaduse for its immunomodulatory activities. It is an approved therapeuticfor the treatment of autoimmune disorders such as immunothrombocytopenia(ITP), chronic inflammatory demyelinating polyneuropathy (CIDP),Kawasaki's disease, and Guillain-Barre syndrome, and is used in agrowing number of autoimmune and inflammatory disorders. Itsanti-inflammatory activity has been shown to result from the presence ofa specific glycan, the α2,6-sialylated, complex bi-antennary structurepresent on the C_(H)2 domain of the Fc, and found in small proportion ofthe heterogenous antibody preparations in IVIG. Sialylation of the Fcglycan on the C_(H)2 domain results in IgGs that can engage type II Fcreceptors such as SIGN-R1, DC-SIGN, and CD23, while reducing theirbinding affinity to type I FcRs. Studies in mouse models of seruminduced arthritis, antibody-dependent ITP, nephrotoxic nephritis, andautoimmune blistering diseases (ABD) confirmed the anti-inflammatoryactivity of the sialylated Fc, whether from IVIG or generated fromrecombinantly expressed IgG1. Moreover, increasing the percentage ofsialylated Fc fragments either in WIG or recombinant expressed IgG1resulted in an enhanced therapeutic potency of these preparations.Elucidation of the mechanism by which sFc induces an anti-inflammatoryresponse was first reported in murine models of arthritis, demonstratingthat selective binding of sialylated Fc to type II FcRs resulted in theproduction of IL-33 by regulatory macrophages, that in turn stimulatedIL-4 secretion from basophils. IL-4 induced the up-regulation of theinhibitory receptor FcγRIIB on effector macrophages thereby increasingthe activation threshold of these cells and suppressing inflammation.Subsequent studies have confirmed that IVIG treatment of humanpopulations resulted in both increased serum IL-33 levels and FcγRIIBexpression on lymphoid and myeloid cells, consistent with the murinedata.

As disclosed herein crystallographic and biophysical studies onsialylated and asialylated IgG Fc fragments have provided insights intothe structural basis for the anti-inflammatory activity of sialylatedFc. Sialylation of the complex, bi-antennary glycan of the IgG Fcresults in increased conformational flexibility of the C_(H)2 domain,thereby sampling the closed conformations of the C_(H)2 domain requiredfor type II FcR binding. In contrast, asialylated Fc structuresuniformly result in open Fc conformations, consistent with their bindingspecificity for type I FcRs. Glycan interactions with amino acidresidues of the C_(H)2 domain are disrupted upon sialylation, providinga basis for the observed conformational changes seen in the proteinstructure and consistent with a model proposed for the bindingspecificity of sialylated Fc for type II FcRs. Based on theseobservations the inventors generated a series of Fc variants, targetingthe amino acids of the C_(H)2 domain that interact with the glycan, withthe goal of determining their impact on type II FcR binding and theresulting anti-inflammatory activity. Both gain and loss of functionmutants were examined in this study. The identification of a gain offunction variant, which could mimic the conformational state induced bysialylation, without requiring this specific carbohydrate modification,can simplify the development of anti-inflammatory IgG Fc for therapeuticuse. The inventors succeeded in identifying a mutation (F241A) predictedto increase mobility of the α1,3-arm, and which replicates theanti-inflammatory activity of sialylated Fc even in the absence ofsialylation. The inventors have characterized this variant, incomparison to sialylated Fc, in both antibody and T cell models ofautoimmune inflammation.

While the basis for WIG protection in antibody-mediated models ofinflammation have been extensively studied, as summarized above, recentstudies have demonstrated that IVIG can also protect in classical Tcell-mediated autoimmune disorders, such as EAE as well as in a model ofairway hyperresponsiveness (AHR). This therapeutic effect of IVIG isproposed to result from the activation and expansion of T_(reg) cellsthus suppressing T cell responses by IFNγ-secreting T_(H)1 andIL-17-secreting T_(H)17 cells. The inventors therefore sought toinvestigate if the T_(reg) cell activation and expansion was also theresult of sialylated Fcs or the F241A variant. Using F241A, sialylatedand asialylated IVIG, the inventors investigated the mechanisms ofaction of their immunomodulatory effects on T_(reg) cell activation andsuppression of T cell-dependent autoimmunity. The inventors demonstratethat the sialylation of IVIG is critically required for T_(reg) cellactivation and expansion and the F241A variant is sufficient to suppressT cell-dependent inflammation in the EAE and experimental colitismodels. Furthermore, both sFc and F241A stimulate the production ofInterleukin-33 (IL-33) that in turn activates T_(reg) cells through theST2 receptor contributing to the suppression of T cell-mediatedautoimmune responses.

As disclosed in the examples below, the inventors present functionaldata demonstrating that specific interactions between the Fc backboneand the Fc glycan influence the effector properties of sialylated IgG.The inventors confirmed that the anti-inflammatory activity of sial Fcdepends strictly on the α2,6-linkage of sialic acid since only α2,6-sialFc, but not α2,3-sial Fc, binds to DC-SIGN and suppressesautoantibody-induced arthritic inflammation. How can different sialicacid linkages on the Fc glycan influence Fc structure? The inventorsdiscovered that the proximity of the sialic acid sugar residue to theprotein backbone may determine how sialic acid interacts with specificamino acid side chains on the Fc. Indeed, molecular modeling suggeststhat only α2,6-linked sialic acid could fit into a groove formed byGlu318 and Lys340 at the Cγ2-Cγ3 interface. To determine if this grooveplays a role in the anti-inflammatory activity of α2,6-sial Fc, theinventors characterized the immunosuppressive functions of sial Fcbearing an E318N point mutation. Interestingly, the E318N α2,6-sial Fcfails to initiate anti-inflammatory pathways associated with WTα2,6-sial Fc, such as induction of IL-33 expression or protection fromarthritic inflammation by adoptive transfer of stimulated DC-SIGN⁺ BMMΦto K/B×N serum-challenged mice. Thus, as with the α2,3-linkage of sialicacid, the inventors propose that the E318N mutation abolished theinteraction between α2,6-linked sialic acid and the Fc backbonenecessary for the Fc to adopt its ‘closed’, anti-inflammatory state.

Fc structures typically resolve the α1,3 arms of the Fc glycan withinthe internal cavity formed by the C_(H)2 domains. By occupying thiscavity, the α1,3 arms stabilize the C_(H)2 domains at a distance apartin the ‘open’ conformation to facilitate binding to type I FcRs. Inorder for the proposed model to be correct, the sialylated α1,3 armshave to move out of this cavity towards the C_(H)2-C_(H)3 interface sothat the terminal sialic acid may contact the E318/K340 groove. Thus, byrepositioning the sialylated α1,3 arms to the C_(H)2-C_(H)3 interface,the C_(H)2 domains may draw closer together to fill the now unoccupiedcavity and form the ‘closed’ conformation. Unlike the α1,6 arm of the Fcglycan, which forms multiple non-covalent interactions with the Fcbackbone, the α1,3 arm forms only one known contact in the absence ofsialylation—the aromatic side chain of F241. However, in the crystalstructure of sial Fc, the only amino acid side chain that contacts theFc glycan to show a significant change in orientation is the ringstructure of F241. The inventors believe that the observed 90° rotationof F241 abrogates the hydrophobic stacking interaction it normally formswith the carbohydrate. The inventors believe this to be structurallyimportant because the disruption of this stabilizing interaction shouldimpart greater degrees of freedom, or mobility, to the sialylated α1,3arms of the Fc glycan. With this greater mobility, the sialylated α1,3arms will sample the space outside of the internal cavity with greaterfrequency to encounter the E318/K340 pocket at the C_(H)2-C_(H)3interface. Consistent with the crucial role of F241 in the structure ofα2,6-sial Fc, the inventors found that an F241A mutation thatspecifically disrupts this protein-sugar contact point recapitulated theanti-inflammatory activity of sial Fc independent of sialylation. Bothα2,6-sial and asial F241A Fc bound to DC-SIGN, induced IL-33 expression,and transferred anti-inflammatory activity with stimulated DC-SIGN⁺ BMMΦto K/B×N serum-challenged mice, recapitulating the anti-inflammatoryactivity of IVIG and sialylated IgG. Recently, reports have beenpublished that question the essential role of Fc sialylation formodulating immune responses. The data, and that of several other groups,have confirmed the anti-inflammatory role of sialylated Fc in multiplemodels of antibody-mediated inflammation. These discrepancies are likelythe result of non-linear dosing of IVIG in selective models and thus notreflective of the physiologically relevant conditions in which IVIG isused.

We have identified that sialylated Fc, as well as variants such asF241A, specifically stimulated iT_(reg) cell expansion and weresufficient to suppress T cell-mediated immune responses in models of EAEand experimental colitis by selective engagement of the type II Fcreceptors, SIGN-R1, or its human orthologue DC-SIGN. Furthermore, theinventors identified IL-33 as an essential mediator of this pathway.IL-33, induced in response to type II FcR engagement by IVIG, sialylatedFc, or F241A, acts pleiotropically, as summarized in FIG. 15. It canmediate IL-4 secretion by basophils to polarize macrophages to an M2phenotype and induce inhibitory FcγRIIB expression, a pathway thatdominates in antibody-mediated autoimmune inflammation, or it can actdirectly on T_(reg) cells to mediate their activation and expansion. Theinventors demonstrated that T_(reg) cells can become activated by thetherapeutic treatment with sialylated Fc that critically relies on thesignaling through the IL-33/ST2 axis. The inventors cannot exclude thepossibility that the IL-33-dependent T_(reg) cell activation is mainlymediated indirectly via dendritic cells as previously described by Mattaand coworkers. However, the inventors did not detect any directinteraction of IVIG with any T cell subset, neither effector nor T_(reg)cells, in contrast to a recently proposed model, nor could the inventorsobserve any evidence in support of WIG providing “Tregitopes”.Specifically, the inventors could detect T_(reg) cell activation andexpansion in experiments in which IVIG- or F241A-treated hDC-SIGN⁺ bonemarrow-derived macrophages have been transferred into naïve recipients.Because the pulsed BMMΦ responded to this treatment with the secretionof IL-33, the data support the conclusion that this cytokine is adominant mediator in the pathway that leads to T_(reg) cell activationand expansion.

Previous studies have established the connection between IL-33 and anamelioration of T cell-mediated inflammation in different mouse modelsthat were always accompanied by an enrichment of T_(reg) cells.Additionally, serum levels of IL-33 have been shown to be highlyelevated upon IVIG administration in human autoimmune patients, thusmaking it a potent inducer of various anti-inflammatory responses. Whenantibodies were used to block ST2 that prevented IL-33 signaling, theinventors observed that this treatment significantly compromised theprotective effect of IVIG/F241A in both a serum transfer arthritismodel, as well as in the EAE model.

It is becoming increasingly clear that far from being a ‘constant’domain, the Fc region of antibodies exhibit heterogeneous structures andfunctions. This invention disclosed herein advances the view that theconformational diversity of the Fc fragment serves as a general strategyfor antibodies to shift receptor specificity in order to effectdifferent immunological outcomes. IgG Fc dynamics are fine-tuned byprotein-glycan interactions, which are in turn regulated by the sugarcomposition of the Fc glycan. The inventors find that a model ofincreased Fc glycan mobility accounts for the biophysical and functionalproperties associated with anti-inflammatory activity of sialylated IgG.Based on these structural and mechanistic observations, the inventorshave developed a surrogate for sialylated IgG, such as F241A, whichoffers the benefit of greater potency and uniformity than IVIG and canbe used for clinical development for both autoantibody- and Tcell-mediated inflammatory diseases.

Polypeptides and Nucleic Acids

Polypeptides

As disclosed herein, this invention provides isolated polypeptideshaving sequences of variants of human IgG Fc that lacks a polysaccharidechain having a terminal sialic acid connected to a galactose moietythrough the α2,6 linkage at the aforementioned Asn²⁹⁷. Suchnon-sialylated IgG Fc variants may be either derived from a naturallyoccurring antibody or expressed in a cell line.

In one embodiment, the Fc region includes one or more substitutions ofthe hIgG1 amino acid sequence. While not limited thereto, exemplary IgG1Fc regions are provided as follows:

Fc of hIgG1 (starting from amino acid 210 in Kabat system):(SEQ ID NO: 1; F241 and F243 are underlined)KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKFc of hIgG1 FA241: (SEQ ID NO: 2)KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVALFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKFc of hIgG1 FA243: (SEQ ID NO: 3)KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

The terms “peptide,” “polypeptide,” and “protein” are used hereininterchangeably to describe the arrangement of amino acid residues in apolymer. A peptide, polypeptide, or protein can be composed of thestandard 20 naturally occurring amino acid, in addition to rare aminoacids and synthetic amino acid analogs. They can be any chain of aminoacids, regardless of length or post-translational modification (forexample, glycosylation or phosphorylation). The peptide, polypeptide, orprotein “of this invention” include recombinantly or syntheticallyproduced versions having the particular domains or portions that bind toDC-SIGN, FcγRIIA, and FcγRIIB The term also encompasses polypeptidesthat have an added amino-terminal methionine (useful for expression inprokaryotic cells).

An “isolated” polypeptide or protein refers to a polypeptide or proteinthat has been separated from other proteins, lipids, and nucleic acidswith which it is naturally associated. The polypeptide/protein canconstitute at least 10% (i.e., any percentage between 10% and 100%,e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%) by dryweight of the purified preparation. Purity can be measured by anyappropriate standard method, for example, by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis. An isolatedpolypeptide/protein described in the invention can be purified from anatural source, produced by recombinant DNA techniques, or by chemicalmethods. A functional equivalent of IgG Fc refers to a polypeptidederivative of IgG Fc, e.g., a protein having one or more pointmutations, insertions, deletions, truncations, a fusion protein, or acombination thereof. It retains substantially the activity of the IgGFc, i.e., the ability to bind to the respective receptor and trigger therespective cellular response. The isolated polypeptide can contain SEQID NO: 2. In general, the functional equivalent is at least 75% (e.g.,any number between 75% and 100%, inclusive, e.g., 75%, 80%, 85%, 90%,95%, and 99%) identical to SEQ ID NO: 2.

The “percent identity” of two amino acid sequences or of two nucleicacids is determined using the algorithm of Karlin and Altschul Proc.Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin andAltschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithmis incorporated into the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength-12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Wheregaps exist between two sequences, Gapped BLAST can be utilized asdescribed in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.

The amino acid composition of the polypeptide described herein may varywithout disrupting the ability of the polypeptide to bind to therespective receptor and trigger the respective cellular response. Forexample, it can contain one or more conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in, e.g.,SEQ ID NO: 2, is preferably replaced with another amino acid residuefrom the same side chain family. Alternatively, mutations can beintroduced randomly along all or part of the sequences, such as bysaturation mutagenesis, and the resultant mutants can be screened forthe ability to bind to the respective receptor and trigger therespective cellular response to identify mutants that retain theactivity as descried below in the examples.

A polypeptide as described in this invention can be obtained as arecombinant polypeptide. To prepare a recombinant polypeptide, a nucleicacid encoding it (e.g., FA241, SEQ ID NO: 2) can be linked to anothernucleic acid encoding a fusion partner, e.g., glutathione-s-transferase(GST), 6×-His epitope tag, or M13 Gene 3 protein. The resultant fusionnucleic acid expresses in suitable host cells a fusion protein that canbe isolated by methods known in the art. The isolated fusion protein canbe further treated, e.g., by enzymatic digestion, to remove the fusionpartner and obtain the recombinant polypeptide of this invention.

Nucleic Acids

Another aspect of the invention features an isolated nucleic acidcomprising a sequence that encodes the polypeptide or protein describedabove. A nucleic acid refers to a DNA molecule (e.g., a cDNA or genomicDNA), an RNA molecule (e.g., an mRNA), or a DNA or RNA analog. A DNA orRNA analog can be synthesized from nucleotide analogs. The nucleic acidmolecule can be single-stranded or double-stranded, but preferably isdouble-stranded DNA. An “isolated nucleic acid” refers to a nucleic acidthe structure of which is not identical to that of any naturallyoccurring nucleic acid or to that of any fragment of a naturallyoccurring genomic nucleic acid. The term therefore covers, for example,(a) a DNA which has the sequence of part of a naturally occurringgenomic DNA molecule but is not flanked by both of the coding sequencesthat flank that part of the molecule in the genome of the organism inwhich it naturally occurs; (b) a nucleic acid incorporated into a vectoror into the genomic DNA of a prokaryote or eukaryote in a manner suchthat the resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein. Thenucleic acid described above can be used to express the fusion proteinof this invention. For this purpose, one can operatively linked thenucleic acid to suitable regulatory sequences to generate an expressionvector.

A vector refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. The vector can becapable of autonomous replication or integrate into a host DNA. Examplesof the vector include a plasmid, cosmid, or viral vector. The vectorincludes a nucleic acid in a form suitable for expression of the nucleicacid in a host cell. Preferably the vector includes one or moreregulatory sequences operatively linked to the nucleic acid sequence tobe expressed.

A “regulatory sequence” includes promoters, enhancers, and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those that direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein or RNA desired, and the like. The expressionvector can be introduced into host cells to produce a polypeptide ofthis invention. A promoter is defined as a DNA sequence that directs RNApolymerase to bind to DNA and initiate RNA synthesis. A strong promoteris one which causes mRNAs to be initiated at high frequency.

Any polynucleotide as mentioned above or a biologically equivalentpolynucleotide available to the artisan for the same intended purposemay be inserted into an appropriate expression vector and linked withother DNA molecules to form “recombinant DNA molecules” expressing thisreceptor. These vectors may be comprised of DNA or RNA; for most cloningpurposes DNA vectors are preferred. Typical vectors include plasmids,modified viruses, bacteriophage and cosmids, yeast artificialchromosomes and other forms of episomal or integrated DNA. It is wellwithin the purview of the artisan to determine an appropriate vector fora particular use.

A variety of mammalian expression vectors may be used to express theabove-mentioned IgG Fcs in mammalian cells. As noted above, expressionvectors can be DNA sequences that are required for the transcription ofcloned DNA and the translation of their mRNAs in an appropriate host.Such vectors can be used to express eukaryotic DNA in a variety of hostssuch as bacteria, blue green algae, plant cells, insect cells and animalcells. Specifically designed vectors allow the shuttling of DNA betweenhosts such as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector should contain: an origin of replicationfor autonomous replication in host cells, selectable markers, a limitednumber of useful restriction enzyme sites, a potential for high copynumber, and active promoters. Expression vectors may include, but arenot limited to, cloning vectors, modified cloning vectors, specificallydesigned plasmids or viruses. Commercially available mammalianexpression vectors which may be suitable, include but are not limitedto, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega),pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolabs),pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo(Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224),pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146),pUCTag (ATCC 37460), and IZD35 (ATCC 37565).

Also within the scope of this invention is a host cell that contains theabove-described nucleic acid. Examples include E. coli cells, insectcells (e.g., using baculovirus expression vectors), yeast cells, ormammalian cells. See e.g., Goeddel, (1990) Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. To producea polypeptide of this invention, one can culture a host cell in a mediumunder conditions permitting expression of the polypeptide encoded by anucleic acid of this invention, and purify the polypeptide from thecultured cell or the medium of the cell. Alternatively, the nucleic acidof this invention can be transcribed and translated in vitro, e.g.,using T7 promoter regulatory sequences and T7 polymerase.

All of naturally occurring IgG Fcs, genetic engineered IgG Fcs, andchemically synthesized IgG Fcs can be used to practice the inventiondisclosed therein. IgG Fc obtained by recombinant DNA technology mayhave the same amino acid sequence as [FA241] SEQ ID NO: 2) or anfunctionally equivalent thereof. The term “IgG Fc” also coverschemically modified versions. Examples of chemically modified IgG Fcinclude IgG Fcs subjected to conformational change, addition or deletionof a sugar chain, and IgG Fc to which a compound such as polyethyleneglycol has been bound.

One can verify the efficacy of a polypeptide/protein thus-made using ananimal model, such as a transgenic mouse, as described below. Anystatistically significant increase in in vivo expression of IL-33basophils or expression of the FcγRIIB receptor on effector macrophagesindicates the polypeptide/protein is a candidate for treating thedisorders mentioned below. In one embodiment, the above described assaysmay based on measurement of a binding to DC-SIGN protein or DC-SIGN⁽⁺⁾cells. The art is replete with various techniques available to theartisan that will be suitable to measuring the ability of a compound toa DC-SIGN or to DC-SIGN⁽⁺⁾ cells and related changes in expression of agene regulated by the DC-SING pathway, such as IL-33. The artisan willbe capable of mixing and matching these various research tools withoutundue experimentation. Once purified and tested by standard methods oraccording to the assays and methods described in the examples below,non-sialylated IgG Fc variants can be included in pharmaceuticalcomposition for treating inflammatory disorders.

As used herein, “antibody” is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity.

As used herein, “antibody fragments”, may comprise a portion of anintact antibody, generally including the antigen binding or variableregion of the intact antibody or the Fc region of an antibody whichretains FcR binding capability. Examples of antibody fragments includelinear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. The antibody fragmentspreferably retain at least part of the hinge and optionally the CH1region of an IgG heavy chain. More preferably, the antibody fragmentsretain the entire constant region of an IgG heavy chain, and include anIgG light chain.

As used herein, the term “Fc fragment” or “Fc region” is used to definea C-terminal region of an immunoglobulin heavy chain. The “Fc region”may be a native sequence Fc region or a variant Fc region. Although theboundaries of the Fc region of an immunoglobulin heavy chain might vary,the human IgG heavy chain Fc region is usually defined to stretch froman amino acid residue at position Cys226, or from Pro230, to thecarboxyl-terminus thereof.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” as appreciated by one of ordinary skill in the art comprisesan amino acid sequence which differs from that of a native sequence Fcregion by virtue of at least one “amino acid modification.” Preferably,the variant Fc region has at least one amino acid substitution comparedto a native sequence Fc region or to the Fc region of a parentpolypeptide, e.g., from about one to about ten amino acid substitutions,and preferably from about one to about five amino acid substitutions ina native sequence Fc region or in the Fc region of the parentpolypeptide. The variant Fc region herein will preferably possess atleast about 75 or 80% homology with a native sequence Fc region and/orwith an Fc region of a parent polypeptide, and more preferably at leastabout 90% homology therewith, more preferably at least about 95%homology therewith, even more preferably, at least about 99% homologytherewith.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one embodiment of theinvention, FcR is a native sequence human FcR. In another embodiment,FcR, including human FcR, binds an IgG antibody (a gamma receptor) andincludes receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors. FcγRII receptors include FcγRIIA (an “activating receptor”)and FcγRIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor FcγRIIA contains an immunoreceptor tyrosine-basedactivation motif (ITAM) in its cytoplasmic domain. Inhibiting receptorFcγRIIB contains an immunoreceptor tyrosine-based inhibition motif(ITIM) in its cytoplasmic domain (see review in Daron, Annu Rev Immunol,15, 203-234 (1997); FcRs are reviewed in Ravetch and Kinet, Annu RevImmunol, 9, 457-92 (1991); Capel et al., Immunomethods, 4, 25-34 (1994);and de Haas et al., J Lab Clin Med, 126, 330-41 (1995), Nimmerjahn andRavetch 2006, Ravetch Fc Receptors in Fundemental Immunology, ed WilliamPaul 5th Ed. each of which is incorporated herein by reference).

The term “native” or “parent” refers to an unmodified polypeptidecomprising an Fc amino acid sequence. The parent polypeptide maycomprise a native sequence Fc region or an Fc region with pre-existingamino acid sequence modifications (such as additions, deletions and/orsubstitutions).

Compositions

Within the scope of this invention is a composition that contains asuitable carrier and one or more of the agents described above, such asthe non-sialylated IgG Fc variants. The composition can be apharmaceutical composition that contains a pharmaceutically acceptablecarrier or a cosmetic composition that contains a cosmeticallyacceptable carrier.

The term “pharmaceutical composition” refers to the combination of anactive agent with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vivo or exvivo. A “pharmaceutically acceptable carrier,” after administered to orupon a subject, does not cause undesirable physiological effects. Thecarrier in the pharmaceutical composition must be “acceptable” also inthe sense that it is compatible with the active ingredient and can becapable of stabilizing it. One or more solubilizing agents can beutilized as pharmaceutical carriers for delivery of an active compound.Examples of a pharmaceutically acceptable carrier include, but are notlimited to, biocompatible vehicles, adjuvants, additives, and diluentsto achieve a composition usable as a dosage form. Examples of othercarriers include colloidal silicon oxide, magnesium stearate, cellulose,and sodium lauryl sulfate.

The above-described composition, in any of the forms described above,can be used for treating disorders characterized by inflammation. Aneffective amount refers to the amount of an active compound/agent thatis required to confer a therapeutic effect on a treated subject.Effective doses will vary, as recognized by those skilled in the art,depending on the types of diseases treated, route of administration,excipient usage, and the possibility of co-usage with other therapeutictreatment.

A pharmaceutical composition of this invention can be administeredparenterally, orally, nasally, rectally, topically, or buccally. Theterm “parenteral” as used herein refers to subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional, or intracranialinjection, as well as any suitable infusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent. Such solutionsinclude, but are not limited to, 1,3-butanediol, mannitol, water,Ringer's solution, and isotonic sodium chloride solution. In addition,fixed oils are conventionally employed as a solvent or suspending medium(e.g., synthetic mono- or diglycerides). Fatty acid, such as, but notlimited to, oleic acid and its glyceride derivatives, are useful in thepreparation of injectables, as are natural pharmaceutically acceptableoils, such as, but not limited to, olive oil or castor oil,polyoxyethylated versions thereof. These oil solutions or suspensionsalso can contain a long chain alcohol diluent or dispersant such as, butnot limited to, carboxymethyl cellulose, or similar dispersing agents.Other commonly used surfactants, such as, but not limited to, TWEENS orSPANS or other similar emulsifying agents or bioavailability enhancers,which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms also can be used for thepurpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include, but are not limited to, lactose and cornstarch. Lubricating agents, such as, but not limited to, magnesiumstearate, also are typically added. For oral administration in a capsuleform, useful diluents include, but are not limited to, lactose and driedcorn starch. When aqueous suspensions or emulsions are administeredorally, the active ingredient can be suspended or dissolved in an oilyphase combined with emulsifying or suspending agents. If desired,certain sweetening, flavoring, or coloring agents can be added.

Pharmaceutical compositions for topical administration according to thedescribed invention can be formulated as solutions, ointments, creams,suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils.Alternatively, topical formulations can be in the form of patches ordressings impregnated with active ingredient(s), which can optionallycomprise one or more excipients or diluents. In some preferredembodiments, the topical formulations include a material that wouldenhance absorption or penetration of the active agent(s) through theskin or other affected areas. The topical composition is useful fortreating inflammatory disorders in the skin, including, but not limitedto eczema, acne, rosacea, psoriasis, contact dermatitis, and reactionsto poison ivy.

A topical composition contains a safe and effective amount of adermatologically acceptable carrier suitable for application to theskin. A “cosmetically acceptable” or “dermatologically-acceptable”composition or component refers a composition or component that issuitable for use in contact with human skin without undue toxicity,incompatibility, instability, allergic response, and the like. Thecarrier enables an active agent and optional component to be deliveredto the skin at an appropriate concentration(s). The carrier thus can actas a diluent, dispersant, solvent, or the like to ensure that the activematerials are applied to and distributed evenly over the selected targetat an appropriate concentration. The carrier can be solid, semi-solid,or liquid. The carrier can be in the form of a lotion, a cream, or agel, in particular one that has a sufficient thickness or yield point toprevent the active materials from sedimenting. The carrier can be inertor possess dermatological benefits. It also should be physically andchemically compatible with the active components described herein, andshould not unduly impair stability, efficacy, or other use benefitsassociated with the composition. The topical composition may be acosmetic or dermatologic product in the form known in the art fortopical or transdermal applications, including solutions, aerosols,creams, gels, patches, ointment, lotion, or foam.

Treatment Methods

The described invention provides methods for treating in a subject aninflammatory disorder. The term “inflammatory disorder” refers to adisorder that is characterized by abnormal or unwanted inflammation,such as an autoimmune disease. Autoimmune diseases are disorderscharacterized by the chronic activation of immune cells undernon-activating conditions. Examples include psoriasis, inflammatorybowel diseases (e.g., Crohn's disease and ulcerative colitis),rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, lupus,type I diabetes, primary biliary cirrhosis, and transplant.

Other examples of inflammatory disorders that can be treated by themethods of this invention include asthma, myocardial infarction, stroke,inflammatory dermatoses (e.g., dermatitis, eczema, atopic dermatitis,allergic contact dermatitis, urticaria, necrotizing vasculitis,cutaneous vasculitis, hypersensitivity vasculitis, eosinophilicmyositis, polymyositis, dermatomyositis, and eosinophilic fasciitis),acute respiratory distress syndrome, fulminant hepatitis,hypersensitivity lung diseases (e.g., hypersensitivity pneumonitis,eosinophilic pneumonia, delayed-type hypersensitivity, interstitial lungdisease (ILD), idiopathic pulmonary fibrosis, and ILD associated withrheumatoid arthritis), and allergic rhinitis. Additional examples alsoinclude myasthenia gravis, juvenile onset diabetes, glomerulonephritis,autoimmune throiditis, ankylosing spondylitis, systemic sclerosis, acuteand chronic inflammatory diseases (e.g., systemic anaphylaxia orhypersensitivity responses, drug allergies, insect sting allergies,allograft rejection, and graft-versus-host disease), and Sjogren'ssyndrome.

In one embodiment, the invention provides methods for treating a Tcell-mediated disease. As used herein a T cell-mediated disease refersto any inflammatory disorder characterized by an abnormal low level ofregulatory T cells (Treg cells) or by abnormally activated Teffectorcells. Examples of this disease include, but are not limited to,multiple sclerosis, Type I diabetes, Myasthenia gravis, rheumatoidarthritis (RA), systemic lupus erythematosus (SLE), and Psoriasis (seee.g., Fundamental Immunology, Paul, W., ed., 7th edition, LippincottWilliams & Wilkins, 2012, p 832).

A “subject” refers to a human and a non-human animal. Examples of anon-human animal include all vertebrates, e.g., mammals, such asnon-human mammals, non-human primates (particularly higher primates),dog, rodent (e.g., mouse or rat), guinea pig, cat, and rabbit, andnon-mammals, such as birds, amphibians, reptiles, etc. In oneembodiment, the subject is a human. In another embodiment, the subjectis an experimental, non-human animal or animal suitable as a diseasemodel.

A subject to be treated for an inflammatory disorder can be identifiedby standard diagnosing techniques for the disorder. Optionally, thesubject can be examined for the level or percentage of one or more ofcytokines or cells a test sample obtained from the subject by methodsknown in the art. If the level or percentage is at or below a thresholdvalue (which can be obtained from a normal subject), the subject is acandidate for treatment described herein. To confirm the inhibition ortreatment, one can evaluate and/or verify the level or percentage of oneor more of the above-mentioned cytokines or cells in the subject aftertreatment.

“Treating” or “treatment” refers to administration of a compound oragent to a subject who has a disorder with the purpose to cure,alleviate, relieve, remedy, delay the onset of, prevent, or amelioratethe disorder, the symptom of the disorder, the disease state secondaryto the disorder, or the predisposition toward the disorder.

An “effective amount” or “therapeutically effective amount” refers to anamount of the compound or agent that is capable of producing a medicallydesirable result in a treated subject. The treatment method can beperformed in vivo or ex vivo, alone or in conjunction with other drugsor therapy. A therapeutically effective amount can be administered inone or more administrations, applications or dosages and is not intendedto be limited to a particular formulation or administration route.

The agent can be administered in vivo or ex vivo, alone orco-administered in conjunction with other drugs or therapy, i.e., acocktail therapy. As used herein, the term “co-administration” or“co-administered” refers to the administration of at least two agents ortherapies to a subject. In some embodiments, the co-administration oftwo or more agents/therapies is concurrent. In other embodiments, afirst agent/therapy is administered prior to a second agent/therapy.Those of skill in the art understand that the formulations and/or routesof administration of the various agents/therapies used may vary.

In an in vivo approach, a compound or agent is administered to asubject. Generally, the compound or agent is suspended in apharmaceutically-acceptable carrier (such as, for example, but notlimited to, physiological saline) and administered orally or byintravenous infusion, or injected or implanted subcutaneously,intramuscularly, intrathecally, intraperitoneally, intrarectally,intravaginally, intranasally, intragastrically, intratracheally, orintrapulmonarily.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100 mg/kg.Variations in the needed dosage are to be expected in view of thevariety of compounds/agents available and the different efficiencies ofvarious routes of administration. For example, oral administration wouldbe expected to require higher dosages than administration by i.v.injection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Encapsulation of the compound in a suitable delivery vehicle(e.g., polymeric microparticles or implantable devices) can increase theefficiency of delivery, particularly for oral delivery.

EXAMPLES Example 1 General Methods and Materials

This example describes general methods and materials used in Examples2-8 below.

Mice

Six-to-ten week old, sex and age matched C57BL/6, SIGN-R1^(−/−), andSIGN-R1^(−/−)hDC-SIGN⁺ mice were used for all experiments in compliancewith federal laws and institutional guidelines approved by TheRockefeller University. SIGN-R1^(−/−) mice were bred to CD11c-hDC-SIGN⁺mice to generate SIGN-R1^(−/−)hDC-SIGN⁺ mice. KRN T cell receptortransgenic mice were on a C57BL/6 background and bred to NOD mice tocreate K/B×N mice (Korganow et al. Immunity 1999, 10(4): 451-461). K/B×Nserum was prepared by collecting blood samples from K/B×N mice. Theserum was separated from blood and pooled and frozen into aliquots forfurther usage. In order to induce serum transfer arthritis, 200 μl ofpooled K/B×N serum was injected intraperitoneally. Severity of arthritiswas scored by clinical examination of the paws. The addition of all fourpaws indices reflected the severity of disease. 0 is unaffected, 1 isswelling of one joint, 2 is swelling of more than one joint, and 3 issevere swelling of the entire paw. In all experiments, groups of 4-5mice were used and means and standard error of the mean (SEM) areplotted in graphs.

Reagent and Treatments

IVIG (Octagam, Octapharma) and F241A (Merck) were used at concentrationsindicated in each figure legend. IVIG-Fc and IVIG-F(ab′)₂ preparationswere prepared by papain digestion of WIG for 2 hours at 37° C.Desialylation of IVIG and F241A was performed by neuraminidasetreatment. 100 mg of antibody preparation was incubated with 700 UnitsNeuraminidase (NEB) for 20 hours at 37° C. Antibodies were purified byProtein-G affinity purification and dialyzed against PBS beforeinjection. Sialic acid contents of all antibody preparations wereverified by SNA lectin blotting using SNA-biotin (Vector laboratories).All IVIG and F241A preparations were administered intravenously (i.v.).

Basophils were depleted by daily i.p. injection with 10 μg anti-FcεRI(MAR-1, eBioscience) or hamster IgG isotype control (Sigma) for 4consecutive days.

T_(reg) cells were depleted by i.p. administration of 400 μg ofanti-CD25 (Setiady et al. Eur J Immunol 2010, 40(3): 780-786) (PC61,BioXcell) 3 days before EAE induction as well as one day after eachIVIG/F241A injection until the end of the experiment.

Blocking of the IL-33 receptor ST2 was achieved by i.v. injections of100 μg of anti-T1/ST2 (DJ8, MD Biosciences) on day 0 as well as everyfifth day post EAE induction.

For cytokine treatment, mice received a single i.v. injection of 2.5 μgof IL-4ic (Peprotech) or 0.5 μg IL-33 i.p. on 4 consecutive days orevery two days in EAE experiments.

IL-6 serum levels were measured by an in vivo cytokine capture assay asdescribed (Finkelman et al. Curr Protoc Immunol 2003, Chapter 6: Unit6.28). IL-33 in cell culture supernatants was detected and measured byELISA, respectively, as suggested by the manufacturer (eBioscience).

Flow Cytometry

In order to analyze lymphocytes and bone marrow cells, single cellsuspensions were prepared from spleens, lymph nodes, and bone marrow.After red blood cell lysis cells were stained with the respectiveantibodies and analyzed using a FACSCalibur (BD Biosciences). Theantibodies used for murine cell stainings were anti-CD4 (GK1.5),anti-Foxp3 (FJK-16s), anti-IFNγ (XMG1.2) from eBiosciences, anti-CD25(PC61), anti-IL-17A (TC11-18H10.1), anti-CD209 (9E9A8), anti-Helios(22F6), anti-CD11c (N418), anti-F4/80 (BM8) from BioLegend andanti-T1/ST2 (MD Biosciences).

Differentiation of Bone Marrow-Derived Macrophages and Transfers

Bone marrow cells were isolated from femurs and tibias, cultured in10-cm plates in DMEM supplemented with 20% fetal bovine serum, 2%penicillin/streptomycin (Invitrogen), 1% L-glutamine 200 mM(Invitrogen), 0.1% β-mercaptoethanol and M-CSF (40 ng/mL, Peprotech) for5-7 days at 37° C. Flow cytometry was used to analyze bonemarrow-derived macrophages (BMMΦ) in the cell cultures (>90% CD11b⁺F4/80⁺ cells). Cells were recovered from plates, washed and seeded infresh tissue culture plates, and subsequently pulsed with PBS, IVIG (15mg/mL) or F241A (80 μg/ml) for 3 hours at 37° C. Cells were thenextensively washed and 1×10⁶ macrophages were administered intravenouslyto recipient mice. One hour post transfer, mice were challenged withK/B×N sera.

Quantitative Real-Time PCR

Total RNA was isolated from bone marrow-derived macrophages using theRNeasy Mini Kit (Qiagen) and reverse transcribed with Superscript IIIReverse Transcriptase (Invitrogen). Quantitative PCR was performed tomeasure IL-33 mRNA levels using a C1000 Touch Thermal Cycler (BioRad)with primer sets for IL-33 (5′-TCACTGCAGGAAAGTACAGCA-3′ (forward, SEQ IDNO: 4) and 5′-AGTAGCACCTGGTCTTGCTC-3′ (reverse, SEQ ID NO: 5)) and Gapdh(5′-ACAGTCCATGCCATCACTGCC-3′ (forward, SEQ ID NO: 6) and5′-GCCTGCTTCACCACCTTCTTG-3′ (reverse, SEQ ID NO: 7)). Gene expressionlevels were calculated by normalization to Gapdh mRNA levels.

In Vitro T_(reg) Cell Differentiation

Single cell suspensions were prepared from spleens and lymph nodes ofnaïve C57BL/6 wild-type mice. Naïve CD4⁺ T cells were isolated bymagnetic cell separation (Miltenyi Biotec) and cultured for 3 days inRPMI supplemented with 10% fetal bovine serum, 2%penicillin/streptomycin (Invitrogen), 1% L-glutamine 200 mM (Invitrogen)and 0.1% β-mercaptoethanol, anti-CD3 (17A2, eBioscience) and anti-CD28(37.51, eBioscience) antibodies in 24-Well cell culture plates that werecoated with anti-Hamster IgG (MP Bio). For T_(reg) cell differentiation,TGF-β (2.5 ng/mL, Peprotech) was added. IL-33 (1 ng/mL, BioLengend) orIL-23 (20 ng/mL Peprotech) was added to the cell cultures as indicatedin the figure legend. On day 3, cells were recovered and T_(reg) cells(CD4⁺ CD25⁺ Foxp3⁺) were analyzed by FACS.

T Cell Transfer Colitis

For T cell transfer colitis, 5×10⁵ naïve CD4⁺ CD45RB^(hi)CD25⁻ T cellsfrom C57BL/6 wild-type animals were sorted and injected i.p. intoC57BL/6 Rag1^(−/−) recipient mice. Body weight loss was measured twice aweek and used as a means of disease severity.

Experimental autoimmune encephalomyelitis Six-to-eight week old miceC57BL/6, SIGN-R1^(−/−), or SIGN-R1^(−/−) hDC-SIGN⁺ mice were immunizedsubcutaneously with 200 μl of an emulsion consisting of 200 μg MOG₃₅₋₅₅peptide (MEVGWYRSPFSRVVHLYRNGK, AnaSpec, SEQ ID NO: 8) emulsified incomplete Freund's adjuvant (Difco Laboratories). On day 0 and 2, micereceived 200 μg of pertussis toxin (List Biological) intraperitoneally.Development of disease was monitored daily according to the followingcriteria: 0—No clinical signs; 0.5—Partially limp tail; 1—Paralyzedtail; 2—Loss in coordinated movement; hind limb paresis; 2.5—One hindlimb paralyzed; 3—Both hind limbs paralyzed; 3.5—Hind limbs paralyzedand hunched back; 4—Severely hunched back and weakness in forelimbs;4.5—Forelimbs paralyzed; 5—Moribund (Stromnes et al. Nat Protoc 2006,1(4): 1810-1819).

Example 2 F241A Mimics Sialylated IgG-Fc and Protects from AutoantibodyInduced Inflammation

The crystal structures of non-sialylated and sialylated IgG moleculesshow differences in the orientation of the heavy chains. Whilenon-sialylated IgG (GOF-form) remains in an open conformation andprovides a structure capable of interacting with type I Fc receptors,the sialylated IgG (G2FS2-form) is more flexible allowing alternateconformations (open and closed) (Ahmed et al. J Mol Biol 2014, 426(18):3166-3179) enabling it to bind to type II Fc receptors (Sondermann etal. Proc Natl Acad Sci USA 2013, 110(24): 9868-9872). As shown in FIG.1a , the aromatic side chains of F241 in an asial Fc structure arestacked with respect to each other (FIG. 1a , left panel). In thisorientation, the phenylalanine side chain forms a hydrophobicinteraction with sugar residues in the α1,3 arm of the Fc glycan.Surprisingly, the aromatic ring of F241 in the sial Fc structureexhibits a near 90° rotation relative to the aromatic ring of F241 inthe asial Fc structure (FIG. 1a , right panel). This suggests that uponsialylation, the interaction between F241 and the α1,3 arm of the Fcglycan may be disrupted, which potentially contributes to the observedchanges in antibody structure and function.

Hence, to mimic this disruption in protein-glycan interaction, theinventors introduced an alanine point mutation at residue F241 (F241A)and determined how this mutation altered the activity of sial and asialFc. The inventors produced α2,6-sialylated F241A Fc by expressing therecombinant protein in 293 cells stably overexpressing theglycosyltransferases ST6GalI and β4GalTI. For comparison, a fraction ofthis sial F241A Fc preparation was subsequently treated withneuraminidase to remove sialic acid yielding asial F241A Fc. Theinventors confirmed the sialylation status of both F241A Fc preparationsby lectin blotting. The inventors next verified that sial F241A Fcretained DC-SIGN binding activity in an ELISA format as demonstrated byincreased receptor binding affinity relative to asial WT Fc (FIG. 8).However, neuraminidase treatment of F241A Fc did not abolish DC-SIGNbinding establishing that the F241A mutation conferred DC-SIGN bindingactivity independent of sialic acid.

To determine if the F241A mutation resulted in functional DC-SIGNbinding and signaling, the inventors investigated the ability of F241Aand sFc to induce IL-33 expression in DC-SIGN expressing bonemarrow-derived macrophages (BMMΦ). Only sFc induces IL-33 expression inthese cells, while both sialylated and asialylated F241A Fc inducedIL-33 expression in BMMΦ in a DC-SIGN-dependent manner (FIG. 1b ).Furthermore, hDC-SIGN⁺ BMMΦ stimulated with asial F241A Fc, as well assFc, suppressed footpad swelling when transferred to mice challengedwith arthritogenic K/B×N serum in a DC-SIGN dependent manner (FIG. 1c ).Furthermore, protection was only achieved with sialylated wild-type Fc,whereas the Fc mutant F241A was capable of protecting mice, even whennon-sialylated (FIG. 1d ). Thus, the F241A mutation recapitulatesseveral Fc functions in assays developed to measure sial Fc activity.

To further define the activity of F241A as an anti-inflammatorymolecule, the inventors challenged C57BL/6 wild-type mice with K/B×Nsera and treated them either with PBS, IL-4ic or asialylated F241A(0.033 g/kg). Serum 1L-6 levels were significantly reduced in mice thatreceived IL-4ic or F241A (FIG. 2a ). Consistent with this observation,IL-4ic- and F241A-treated mice showed reduced clinical signs ofarthritis (FIG. 2b ), showing that F241A is sufficient to suppressinflammation comparable to IVIG and sFc (Kaneko et al. Science 2006,313(5787): 670-673, and Anthony et al. Science 2008, 320(5874):373-376).

To confirm that this suppression by F241A is type II FcR dependent, theinventors used SIGN-R1^(−/−) or SIGN-R1^(−/−) hDC-SIGN⁺ recipients. Micereceived either sialylated wild-type Fc or neuraminidase-treatednon-sialylated F241A (both 0.033 g/kg) and were challenged with K/B×Nsera. Suppression of arthritic inflammation was achieved by bothpreparations; however, only mice that expressed the BAC transgene humanDC-SIGN (hDC-SIGN⁺) were protected (FIG. 2c ), demonstrating that thepresence of the type II Fc receptor SIGN-R1 or its human orthologueDC-SIGN, respectively, is required for the immunomodulatory effectinduced by sialylated Fc and F241A.

Sialic acid can be linked to the penultimate galactose of the complex,bi-antennary Fc glycan in either α2,3, α2,6 or α2,8 conformations. Theinventors have previously reported that only the α2,6-linked glycoformof sialic acid is biologically active (Anthony et al. Science 2008,320(5874): 373-376). Previous modeling data on the structural analysisof different Fc sialoforms (Sondermann et al. Proc Natl Acad Sci USA2013, 110(24): 9868-9872) predicted that the Glu318/Lys340 pocket at theCγ2-Cγ3 interface was required for the biological activity of α2,6-sialFc and could uniquely accommodate this glycoform, while the α2,3 linkedsialic acid would be sterically inhibited from fitting into this pocket.To test this prediction, the inventors introduced an E318N pointmutation into IgG1 Fc and compared its properties, when α2,6 sialylated,to wild-type α2,6-sial Fc. While comparable degrees of sialylation wereachieved with mutant as compared to the wild-type, only the wild-typesialylated Fc was capable of stimulating IL-33 expression in hDC-SIGN⁺BMMΦ (FIG. 9a ). Mice receiving BMMΦ stimulated with α2,6-sial wild-typeFc, but not α2,6-sial E318N Fc, exhibited reduced clinical signs ofdisease (FIG. 9b ), as well as lower levels of IL-6 (FIG. 3c ). Togetherthese results define both F241 and E318 as residues that contribute tothe anti-inflammatory activity of α2,6-sial Fc.

Example 3 Sialylated Fc/F241A Activates Treg Cells

Recent studies in patients and in animal models suggest thatadministration of WIG can result in an expansion of T_(reg) cells(Ephrem et al. Blood 2008, 111(2): 715-722, and Bayry J et al.Rheumatol, vol. 39: Canada, 2012, pp 450-451), effectively dampening Tcell-dependent autoimmune reactions by increasing the number and thesuppressive capacity of T_(reg) cells (Kessel et al. J Immunol 2007,179(8): 5571-5575). To determine which component of an IVIG preparationmay be responsible for this effect, the inventors used IVIG (1 g/kg),F(ab′)₂ (0.66 g/kg) or Fc (0.33 g/kg) preparations of IVIG andadministered them intravenously at equimolar concentrations into C57BL/6wild-type mice. Four days post injection the inventors analyzed thepercentage of splenic CD4⁺CD25⁺Foxp3⁺ T_(reg) cells by flow cytometry.In comparison to PBS-treated mice, administration of intact IVIG or itsFc fragments led to a two-fold expansion of T_(reg) cells and wasabrogated by using IVIG-F(ab′)₂ (FIG. 3a ).

We next determined the role of Fc sialylation in T_(reg) cell expansionin an ongoing inflammatory response. K/B×N-challenged C57BL/6 mice weretreated either with PBS, IVIG (1 g/kg), neuraminidase-treatednon-sialylated IVIG (NA-IVIG)(1 g/kg) or F241A (0.033 g/kg) andevaluated for disease progression and T_(reg) cell expansion. Asobserved previously (Schwab et al. Eur J Immunol 2014, 44(5): 1444-1453,and Anthony et al. Nature 2011, 475(7354): 110-113), clinical scores ofarthritis showed that IVIG and F241A, but not non-sialylated IVIG,protected mice from arthritis (FIG. 3b ). T_(reg) cell expansion wasobserved in IVIG and F241A treated mice, but not in asial-IVIG treatedmice (FIG. 3c ) suggesting that the T_(reg) cell subset becomesselectively expanded by sialylated Fc and F241A, respectively.

Example 4 IVIG-/F241A-Activated T Cells Suppress CD4⁺ T Cell Responses

To evaluate whether T_(reg) cell expansion in response to IVIG/F241A, isable to suppress CD4⁺ T cell responses, the inventors induced EAE inC57BL/6 wild-type mice by immunization with MOG₃₅₋₅₅ peptide emulsifiedin CFA. Five days post induction the mice were treated with either PBS,IVIG or NA-IVIG (both 1 g/kg) to discriminate between effectsspecifically triggered by sialylated IgG. Clinical scores of EAE showedthat mice that received IVIG had significantly reduced clinical scoreswhen compared to PBS-treated mice (FIG. 4a ). However, when asial-IVIG(NA-IVIG) was administered, the protective effect was abolished. Todetermine the potential mechanistic basis for this effect, the inventorscharacterized cells from draining lymph nodes from treated animals andanalyzed the percentages of CD4⁺ effector T cells. All subpopulations ofCD4⁺ effector T cells, T_(H)1 (IFN-γ⁺), T_(H)17 (IL-17A⁺), and IFN-γ⁺IL-17A⁺ CD4⁺ T cells were present at similar percentages (FIG. 4b );however, the percentages of CD4⁺CD25⁺Foxp3⁺ T_(reg) cells weresignificantly elevated in mice that were given IVIG compared to PBS- orNA-IVIG-treated groups (FIG. 4c ).

To assess whether protection from disease is specifically mediatedthrough activation and expansion of T_(reg) cells, the inventors testedthe protective potential of F241A as a surrogate for IVIG in untreatedand T_(reg) cell depleted mice. T_(reg) cell depletion was achieved byadministration of an anti-CD25 antibody (PC61). Mice treated with F241A(0.033 g/kg) alone were protected, as compared to PBS-treated mice (FIG.4d ), which showed that F241A was equally potent in protecting from Tcell responses as IVIG. However in T_(reg) cell depleted mice, thisprotective effect was significantly reduced. FACS analysis indicatedthat neither F241A nor PC61 treatment significantly affected CD4⁺effector T cells (FIG. 4e ), whereas T_(reg) cell levels were reduced byPC61 treatment (FIG. 4f ). Depletion of T_(reg) cells thus correlatedwith the loss of protection from EAE observed in F241A-treated animals.To distinguish between natural (nT_(reg)) and inducible T_(reg)(iT_(reg)) cells, the inventors analyzed the T_(reg) cells in IVIG- andPBS-treated EAE mice for their expression of the nTreg-specifictranscription factor Helios (Thornton et al. J Immunol 2010, 184(7):3433-3441). The expansion of T_(reg) cells did not correlate with anincrease in Helios expression (FIG. 10c ), which indicates that IVIG (1g/kg), which clearly protected mice from EAE (FIGS. 10 a and b),specifically induces CD4⁺CD25⁺Foxp3⁺Helios⁻ inducible T_(reg) (iT_(reg))cells.

Together these results indicate that sialylated IVIG as well as F241Alead to the activation and expansion of T_(reg) cells resulting in thesuppression of CD4⁺ effector T cell responses and clinical disease inEAE.

Example 5 Type II Fc Receptors are Required for sFc-Mediated Protectionfrom EAE

The requirement of the type II Fc receptors SIGN-R1 and hDC-SIGN forsFc-induced suppression of inflammation has been extensively studied inthe context of autoantibody-mediated diseases and for the stimulation ofIL-33 production (Anthony et al. Nature 2011, 475(7354): 110-113). Theinventors therefore examined the requirement for the type II Fcreceptor, SIGN-R1, for sFc-mediated T_(reg) cell stimulation. EAE wasinduced in C57BL/6 wild-type or SIGN-R1^(−/−) mice and then treated withIVIG (1 g/kg) or PBS as control. While wild-type mice were againprotected from EAE by IVIG, this protective effect was significantlyreduced in SIGN-R1 knockout mice (FIG. 5a ). This is consistent with theobservations that in the SIGN-R1^(−/−) background IVIG (1 g/kg) neitherprotects from K/B×N-induced arthritis (FIG. 11a ) nor induced T_(reg)cell activation (FIG. 11b ). By contrast, when IL-33 levels werereconstituted by administration of exogenous IL-33, SIGN-R1^(−/−) micewere partially protected from EAE (FIG. 5b ), which was associated withincreased T_(reg) cell numbers (data not shown). Similarly, transgeneexpression of hDC-SIGN complements the loss of SIGN-R1 (Anthony et al.Nature 2011, 475(7354): 110-113) and results in reduced EAE clinicalscores in both IVIG (1 g/kg) and F241A (0.033 g/kg) treated mice (FIG. 5c and d).

Example 6 IL-33 is a Critical Mediator of sFc-Triggered T_(reg) CellActivation

Up-regulation of FcγRIIB on effector macrophages by sialylated Fccritically depends on production and secretion of the alarmin IL-33(Anthony et al. Nature 2011, 475(7354): 110-113). Moreover recentfindings indicated that IL-33 itself has a positive effect on T_(reg)cell stimulation and activation (Turnquist et al. J Immunol 2011,187(9): 4598-4610, and Matta et al. J Immunol 2014, 193(8): 4010-4020)and thereby contributes to the suppression of inflammation in a mousemodel of experimental colitis (Schiering et al. Nature 2014, 513(7519):564-568). To test the possibility that sFc-induced production of IL-33may also contribute to T_(reg) cell stimulation, the inventors observednaïve CD4⁺ T cells that were isolated from C57BL/6 wild-type mice andcultured for three days in the presence of anti-CD3, anti-CD28antibodies and TGF-β to specifically drive T_(reg) cell differentiation.The cells were either left untreated or treated in combinations withIL-33 and IL-23. Flow cytometric analysis of the percentages of CD4⁺Foxp3⁺ T_(reg) cells in the cultures showed that addition of IL-33 had asynergistic effect on T_(reg) cell differentiation as well as on Foxp3expression (FIG. 12) as was previously reported by Schiering andcoworkers (Schiering et al. Nature 2014, 513(7519): 564-568). MoreoverIL-33 induced up-regulation of the IL-33 receptor ST2 on T_(reg) cells.Addition of IL-23 to the T_(reg) cell culture counteracted the effect ofIL-33 (FIG. 12), consistent with IL-23 being a negative regulator of ST2(Schiering et al. Nature 2014, 513(7519): 564-568, and Izcue et al.Immunity 2008, 28(4): 559-570).

Next the inventors determined whether administration of IL-33 alsoaffects T_(reg) cells in vivo. IL-33 (0.5 μg) was given to C57BL/6wild-type mice daily for four consecutive days. On day five, spleenswere analyzed for T_(reg) cell numbers. IL-33 administration resulted ina significant increase of T_(reg) cells compared to PBS-treated controlmice (FIGS. 6 a and b). hDC-SIGN⁺ BMMΦ were pulsed either with PBS,IVIG, or non-sialylated F241A, and IL-33 expression was measured byquantitative rtPCR, showing that IVIG and F241A clearly induced IL-33expression in a DC-SIGN-dependent manner (FIG. 6c ). After treatment,BMMΦ were subsequently transferred into C57BL/6 wild-type mice. Fivedays post cell transfer, only mice that received IVIG- or F241A-pulsedhDC-SIGN⁺ BMMΦ had significantly higher levels of T_(reg) cells (FIGS.6d and e ). This phenotype correlated with enhanced expression of theIL-33 receptor ST2 on these cells (FIG. 6d ). In addition, IL-33treatment of EAE resulted in a significant amelioration of EAE symptoms,which correlated with an increase of T_(reg) cells in draining lymphnodes (FIGS. 6 f and g).

Since FcγRIIB up-regulation on effector macrophages has beendemonstrated to require the presence of basophils (Anthony et al. Nature2011, 475(7354): 110-113), the inventors investigated whether basophilsalso play a role in the sFc-mediated T_(reg) cell activation pathway.Mice were treated with PBS or WIG (1 g/kg) and challenged with K/B×Nserum. Mice were also treated with an anti-FcεRI antibody for depletionof basophils or with an isotype control (Sokol et al. Nat Immunol 2008,9(3): 310-318). While basophil depletion (FIG. 13a ), as the inventorshave previously shown (Anthony et al. Nature 2011, 475(7354): 110-113),disrupted the protective effect of IVIG in the K/B×N arthritis model(FIG. 13b ), the ability of IVIG to expand T_(reg) cells was notaffected (FIG. 13c ). This indicates that IL-33 is required for T_(reg)cell activation; however, basophils do not contribute in this pathway.

Example 7 sFc/F241A Activates Inducible T Cells Via the IL-33/ST2 Axis

To further explore the mechanism of T_(reg) cell expansion andactivation in response to sFc and type II FcRs engagement, the inventorsfocused on the role of the IL-33 receptor ST2. As it has recently beenreported, K/B×N-challenged mice treated with PBS or IVIG (1 g/kg) eitheralone or in combination with a ST2 blocking antibody indicated thatblocking the IL-33 receptors reduced the protective effect of IVIG inthe serum transfer arthritis model (Anthony et al. Nature 2011,475(7354): 110-113) (FIG. 7a ). FACS analysis of the T_(reg) cellnumbers in these mice revealed that inhibition of IL-33 signaling alsoabrogated the expansion of T_(reg) cells (FIG. 7b ). Similar resultswere observed in the EAE model, where the therapeutic effect of F241A(0.033 g/kg) was significantly reduced by blocking the IL-33 receptor(FIG. 7c ) with a concomitant reduction in T_(reg) cell expansion (FIG.7d ).

Finally, to determine the generality of the observations on the effectof sFc on T cell-mediated diseases, the inventors used the experimentalcolitis mouse model and treated these mice weekly, starting four weekspost T cell transfer, with IVIG (1 g/kg) or PBS control until the end ofthe experiment. Body weight loss was used as a measure of diseaseseverity, which showed that IVIG-treatment mediated protection (FIGS. 14a, c, and d), and was again accompanied by a significant enrichment ofT_(reg) cells (FIG. 14b ), while CD4⁺ effector T cell levels werecomparable in PBS- and IVIG-treated groups. Based on the fact that onlyCD4⁺CD25⁻ T cells have been adoptively transferred to induce acutecolitis, the IVIG-expanded T_(reg) cells in these mice originated fromperipheral CD4⁺ T cells and are thus iT_(reg) cells.

The foregoing example and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. All publications cited herein arehereby incorporated by reference in their entirety. As will be readilyappreciated, numerous variations and combinations of the features setforth above can be utilized without departing from the present inventionas set forth in the claims. Such variations are not regarded as adeparture from the scope of the invention, and all such variations areintended to be included within the scope of the following claims.

1. A method of increasing a level of regulatory T (T_(reg)) cells in asubject in need thereof, comprising administering to the subject aneffective amount of (i) a first isolated polypeptide comprising an IgGFc region that is sialylated; or (ii) a second isolated polypeptidecomprising a modified sequence that is at least 75% identical to the IgGFc region or a nucleic acid encoding the second polypeptide, wherein themodified sequence has a FA241 mutation.
 2. The method of claim 1,wherein the IgG region in the first isolated polypeptide is sialylatedat a level higher than that of IgG in the subject.
 3. The method ofclaim 1, wherein the subject has an inflammatory disease.
 4. The methodof claim 3, wherein the inflammatory disease is an autoimmune disease.5. The method of claim 4, wherein the autoimmune disease is a Tcell-mediated autoimmune disease.
 6. The method of claim 5, wherein theT cell-mediated autoimmune disease is selected from the group consistingof multiple sclerosis and type I diabetes.
 7. The method of claim 1,wherein the IgG Fc region comprises the sequence of SEQ ID NO:
 1. 8. Themethod of claim 1, wherein the first or second isolated polypeptide hasan ability to bind to DC-SIGN, hFcγRIIA, or hFcγRIIB.
 9. The method ofclaim 8, wherein the isolated polypeptide has an ability to bind tohFcγRIIA or hFcγRIIB at a K_(D) of 2×10⁻⁵ M or lower (i.e., K_(A) of5.0×10⁴ M⁻¹ or higher).
 10. The method of claim 1, wherein the modifiedsequence is (a) substantially free of sialylation or (b) sialylated at alevel lower than that of IgG of the subject.
 11. The method of claim 1,wherein the modified sequence is at least 75% identical to SEQ ID NO: 2.12. A method of treating a T cell-mediated autoimmune disease in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of (i) a first isolated polypeptidecomprising an IgG Fc region that is sialylated; or (ii) a secondisolated polypeptide comprising a modified sequence that is at least 75%identical to the IgG Fc region or a nucleic acid encoding the secondpolypeptide, wherein the modified sequence has a FA241 mutation.
 13. Themethod of claim 12, wherein the IgG region in the first isolatedpolypeptide is sialylated at a level higher than that of IgG in thesubject.
 14. The method of claim 12, wherein the T cell-mediatedautoimmune disease is selected from the group consisting of multiplesclerosis and type I diabetes.
 15. The method of claim 12, wherein theIgG Fc region comprises the sequence of SEQ ID NO:
 1. 16. The method ofclaim 12, wherein the first or second isolated polypeptide has anability to bind to DC-SIGN, hFcγRIIA, or hFcγRIIB.
 17. The method ofclaim 16, wherein the isolated polypeptide has an ability to bind tohFcγRIIA or hFcγRIIB at a K_(D) of 2×10⁻⁵ M or lower (i.e., K_(A) of5.0×10⁴ M⁻¹ or higher).
 18. The method of claim 12, wherein the modifiedsequence is (a) substantially free of sialylation or (b) sialylated at alevel lower than that of IgG of the subject.
 19. The method of claim 12,wherein the modified sequence is at least 75% identical to SEQ ID NO: 2.